Implementation of a Rapid RT-LAMP Saliva-based SARS-CoV-2 Testing Program in the Workplace

Rising SARS-CoV-2 cases, testing delays and the risk of pre-symptomatic and asymptomatic transmission provided the impetus for an in-house rapid testing pro-gram. Employees and their household contacts were encouraged to self-collect saliva samples which were pooled for routine testing using an established colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. In brief, individual or a maximum of four saliva samples were pooled, heat-inactivated to render microorganisms, especially SARS-CoV-2, non-infectious prior to being added to RT-LAMP assay tubes containing either human sample control gene, RNase P or a region of the SARS-CoV-2 gene, ORF1ab. During the second wave of SARS-CoV-2 infections in November 2020, two samples from an employee and a member of their household tested positive via RT-LAMP within two days of each other. A delayed clinical qRT-PCR test confirmation of both individuals 5 days later underscores the power of routine rapid testing with within-the-hour turnaround times. Workplace rapid testing programs using RT-LAMP are flexible in their design, have a reduced cost compared to qRT-PCR, may involve non-invasive self-saliva collection for increased safety for the testing personnel, and can be performed with minimal training.

Serumal and Salivary 25(OH)D and 1,25(OH)D Levels of Head and Neck Cancer Patients

BACKGROUND: Saliva has been suggested as a substitute of serum for the detection of 25 Dihydroxyvitamin D (25(OH)D) in healthy people. However, investigation of salivary 1,25(OH)D has not been clearly reported. Vitamin plays important roles in inhibiting cancer progression. Current study was conducted to investigate serumal and salivary 25(OH)D) and 1,25(OH)D levels of healthy and head and neck cancer (HNC) subjects.METHODS: Research were conducted at Haji Adam Malik Hospital, Medan, Indonesia. Forty HNC and 40 healthy subjects were recruited and selected based on inclusion and exclusion criteria. Medical records were documented, followed by anthropometric evaluation and serum and saliva collection. Laboratory investigation for 25(OH)D and 1,25(OH) was performed using Enzyme-linked immunosorbent assay (ELISA) methods.RESULTS: Significant serumal (p=0.002) and salivary (p=0.016) 25(OH)D mean level differences of HNC and normal groups were obtained. More serumal or salivary 25(OH)D deficient subjects were found in control group than those in HNC group. Meanwhile, serumal and salivary 1,25(OH)D mean levels of HNC group were not significantly different with the ones of control group. There were significant correlations of serumal-salivary 25(OH)D as well as serumal-salivary 1,25(OH)D levels in normal group.CONCLUSION: Serumal and salivary 25(OH)D and 1,25(OH)D levels of HNC group were relatively normal. Salivary 25(OH)D and 1,25(OH)D could be suggested as substitutes for serumal ones.KEYWORDS: vitamin D, 25(OH)D, 1,25(OH)D, head and neck cancer

Diagnostic utility of saliva and its implication in detection of Covid-19 and other diseases

Saliva is a valuable tool for early detection, better treatment, and a better prognosis. Early detection of illnesses is sometimes challenging, and it necessitates additional clinical and laboratory tests, which can delay treatment and have a significant impact on prognosis. A large range of chemicals may be found in saliva, providing useful information for clinical diagnostic purposes.The coronavirus disease pandemic (Covid-19) is the world's largest challenge and global health disaster since World War II. Controlling the epidemic in the community and in hospitals requires a quick and precise diagnosis of Covid-19. For Covid-19 diagnostic testing, nasopharyngeal and oropharyngeal swabs are the suggested specimen types.The collection of these specimens necessitates intimate contact between healthcare staff and patients, which increases the risk of viral transmission. As a result, nasopharyngeal or oropharyngeal swabs are not recommended for sequential viral load monitoring. Saliva specimens are simply collected by having the patient spit into a sterile container. Saliva collection is non-invasive and significantly reduces healthcare personnel' exposure to Covid-19. To develop quick chair side assays for the detection of Covid-19, more study is needed to investigate the potential diagnostic of Covid-19 in saliva.

Food for thought: Eating before saliva collection and interference with SARS-CoV-2 detection

BackgroundSaliva is an optimal specimen for detection of viruses that cause upper respiratory infections including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to its cost-effectiveness and non-invasive collection. However, together with intrinsic enzymes and oral microbiota, children’s unique dietary habits may introduce substances that interfere with diagnostic testing.MethodsTo determine whether children’s dietary choices impact SARS-CoV-2 detection in saliva, we performed a diagnostic study that simulates testing of real-life specimens provided from healthy children (n=5) who self-collected saliva at home before and at 0, 20, and 60 minutes after eating from 20 foods they selected. Each of seventy-two specimens was split into two volumes and spiked with SARS-CoV-2-negative or -positive standards prior to side-by-side testing by reverse-transcription polymerase chain reaction matrix-assisted laser desorption ionization time-of-flight (RT-PCR/MALDI-TOF) assay.ResultsDetection of internal extraction control and SARS-CoV-2 nucleic acids was reduced in replicates of saliva collected at 0 minutes after eating 11 of 20 foods. Interference resolved at 20 and 60 minutes after eating all foods except hot dog in one participant. This represented a significant improvement in detection of nucleic acids compared to saliva collected at 0 minutes after eating (P=0.0005).ConclusionsWe demonstrate successful detection of viral nucleic acids in saliva self-collected by children before and after eating a variety of foods. Fasting is not required before saliva collection for SARS-CoV-2 testing by RT-PCR/MALDI-TOF, but waiting 20 minutes after eating is sufficient for accurate testing. These findings should be considered for SARS-CoV-2 testing and broader viral diagnostics in saliva specimens.

Unstimulated Parotid Saliva Is a Better Method for Blood Glucose Prediction

Objective: Saliva glucose has been widely used in diagnosing and monitoring diabetes, but the saliva collection method will affect saliva glucose concentration. So, this study aims to identify the ideal saliva collection method. Method: A total amount of six saliva collection methods were employed in 80 healthy participants in the morning. Besides, three unstimulated saliva methods were employed in another 30 healthy participants in the morning; in the meantime the blood glucose of these 30 participants was detected with a Roche blood glucose meter. The glucose oxidase method with 2, 4, 6-tribromo-3-hydroxybenzoic acid (TBHBA) as the chromogen has been improved to be suitable for healthy people, through the selection of the optimal pH value and ionic strength of the reaction system. This method was used for the detection of saliva glucose. Results: The improved method obtained absorbance at the wavelength of 520 nm, and the optimized parameter combination was pH 6.5 and 5 mg/dL NaCl. The lower limit of glucose detection was 0.1 mg/dL. Unstimulated saliva glucose concentration was higher than stimulated saliva glucose concentration. Unstimulated parotid saliva glucose concentration was the highest. Besides, unstimulated saliva glucose has a better normal distribution effect. Meantime, it was found that unstimulated parotid saliva was the most highly correlated with blood glucose (R2 = 0.707). Conclusions: the saliva collection method was an important factor that affected saliva glucose concentration. Unstimulated parotid saliva was the most highly correlated with blood glucose, which provided a reference for prediction of diabetes mellitus.

Feasibility of In-home Saliva Collection of Cortisol and DHEA-S as a Biomarker of Stress in Dementia Care Dyads

Dementia afflicts affected individuals and their family caregivers worldwide. Although a non-pharmacological intervention has been recommended as a first-line approach to minimize adverse outcomes (e.g., stress) in dementia care dyads (persons with dementia [PWD] and their family caregivers), most evaluations of such interventions have relied on subjective (e.g., self- or proxy-report) rather than objective (e.g., biomarkers) measures. We aimed to explore the feasibility of saliva collection of cortisol and dehydroepiandrosterone sulfate (DHEA-S) as a non-intrusive method in dementia care dyads. Dementia care dyads living at home were recruited from the memory center in Sweden. Prior to the saliva collection, participants received a one-hour education session with a hands-on demonstration led by a trained study coordinator. Participants were instructed to collect saliva three times (two for morning, one for evening)/day, five days/week for eight consecutive weeks. Out of 32 care dyads (32 PWD and 32 family caregivers), 24 (75.0%) completed the saliva collection. On average, 105.5 (87.92%) and 105.9 (88.25%) samples were collected from PWD and family caregivers during eight weeks. There were no statistically significant differences (p>0.05) in the average number of saliva samples (i.e., total samples, morning or evening samples) between PWD and family caregivers. The findings of this pilot study showed that saliva collection of cortisol and DHEA-S as a stress measurement was feasible in dementia care dyads living at home. Robust and person-centered procedures, tailored educational materials, and effective communication with dementia care dyads should be considered in future biomarker research on stress in dementia care dyads.

Quantitative serology for SARS-CoV-2 using self-collected saliva and finger-stick blood

Convenient and widespread serology testing may alter the trajectory of the COVID-19 pandemic. This study seeks to leverage high-throughput, multiplexed serologic assays, which have been adopted as benchmarks for vaccine efficacy, to support large-scale surveys of SARS-CoV-2 immunity using finger-stick blood and/or saliva. Specifically, we optimized MSD’s serology assays, which were analytically validated for serum, to test self-collected finger-stick blood and saliva samples. We show that these assays can be used with FDA-registered specimen collection devices to obtain quantitative measurements for self-collected samples. Antibody levels were measured using an electrochemiluminescent (ECL) multiplex immunoassay, which has been used to measure humoral responses to several COVID-19 vaccines, including those funded by the U.S. Government’s Operation Warp Speed. First, we show that salivary antibodies are stable without refrigeration or preservatives for at least five days. Using matched samples, we show that testing of saliva and finger-stick blood equivalently identified individuals with humoral responses to CoV-2 antigens. Moreover, we piloted a simple saliva collection kit that can be used to safely send samples through the mail. This work demonstrates that robust methods for self-collection of finger-stick blood and saliva, in combination with quantitative, automated immunoassays, provide the technical capabilities needed to support large-scale serology testing.

Assessing saliva microbiome collection and processing methods

The oral microbiome has been connected with lung health and may be of significance in the progression of SARS-CoV-2 infection. Saliva-based SARS-CoV-2 tests provide the opportunity to leverage stored samples for assessing the oral microbiome. However, these collection kits have not been tested for their accuracy in measuring the oral microbiome. Saliva is highly enriched with human DNA and reducing it prior to shotgun sequencing may increase the depth of bacterial reads. We examined both the effect of saliva collection method and sequence processing on measurement of microbiome depth and diversity by 16S rRNA gene amplicon and shotgun metagenomics. We collected 56 samples from 22 subjects. Each subject provided saliva samples with and without preservative, and a subset provided a second set of samples the following day. 16S rRNA gene (V4) sequencing was performed on all samples, and shotgun metagenomics was performed on a subset of samples collected with preservative with and without human DNA depletion before sequencing. We observed that the beta diversity distances within subjects over time was smaller than between unrelated subjects, and distances within subjects were smaller in samples collected with preservative. Samples collected with preservative had higher alpha diversity measuring both richness and evenness. Human DNA depletion before extraction and shotgun sequencing yielded higher total and relative reads mapping to bacterial sequences. We conclude that collecting saliva with preservative may provide more consistent measures of the oral microbiome and depleting human DNA increases yield of bacterial sequences.

Evaluation of a Modified Bit Device to Obtain Saliva Samples from Horses

(1) Background: Accounting for the well-being of equine partners is a responsibility of those engaged in Equine-Assisted Services (EAS). Researchers took heed of this call to action by developing an innovative way to collect data to assess the physiological indicators of stress in equine participants. The collection of saliva is considered to be a minimally invasive method of data collection and is typically performed using a cotton swab; however, in equines, the introduction of a foreign object may induce stress; (2) Methods: Researchers used a modified bit to collect pooled saliva in an effort to further reduce stress during the saliva collection process. Additionally, the collection of pooled saliva, via the bit, increases the opportunity to consider additional analyses, such as oxytocin, which is more reliable in pooled saliva than site-specific saliva captured with a swab; (3) Results: A data analysis demonstrated that ample saliva was captured using the modified bit. Observational data supported that the horses demonstrated fewer physical stress signals to the bit than to the swab. Thus, the modified bit is a feasible and valid method for equine salivary sample collection; (4) Conclusions: The results suggest that the modified bit provides a viable method to collect equine saliva and supports national calls to prioritize animal welfare analysis, specifically for horses used within EAS. Future research should enhance methodological rigor, including in the process and timing, thereby contributing to the bit’s validation.

Potential of Salivary Biomarkers in Autism Research: A Systematic Review

The diagnostic process for autism spectrum disorders (ASD) is based on a behavioral analysis of the suspected individual. Despite intensive research, no specific and valid biomarker has been identified for ASD, but saliva, with its advantages such as non-invasive collection, could serve as a suitable alternative to other body fluids. As a source of nucleic acid of both human and microbial origin, protein and non-protein molecules, saliva offers a complex view on the current state of the organism. Additionally, the use of salivary markers seems to be less complicated not only for ASD screening but also for revealing the etiopathogenesis of ASD, since enrolling neurotypical counterparts willing to participate in studies may be more feasible. The aim of the presented review is to provide an overview of the current research performed on saliva in relation to ASD, mutual complementing, and discrepancies that result in difficulties applying the observed markers in clinical practice. We emphasize the methodological limitations of saliva collection and processing as well as the lack of information regarding ASD diagnosis, which is critically discussed.